Methylcrotonic acid and derivatives in carbomycin a fermentation

ABSTRACT

Addition of Beta -methylcrotonic acid, or derivatives thereof, to carbomycin A-producing fermentation media gives rise to an increased yield of the antibiotic.

United States Patent Inventor App1. No.

Joseph L. Sardines Gales Ferry, Conn. 805,019

Mar. 6, 1969 Sept. 2 l 1971 Pfizer Inc.

New York, NY.

fi-METHYLCROTONIC ACID AND DERIVATIVES IN CARBOMYCIN A FERMENTATION 6Claims, No Drawings US. Cl 195/114, 195/80 Int. Cl Cl2d 9/00 [50] FieldofSearch 195/114, 117, 80

[56] References Cited UNiTED STATES PATENTS 2,796,379 6/1957 Tanner etal 195/80 X 2,918,413 12/1959 Redemann 195/114 X 3,483,088 12/1969Seeley 195/114 Primary Examiner-Joseph M. Golian Attorney-Connolly &Hutz ABSTRACT: Addition of B-methylcrotonic acid, or derivativesthereof, to carbomycin A-producing fermentation media gives rise to anincreased yield ofthe antibiotic.

B-METHYECRQILQNIQ ACID AND DERIVATIVES IN CARBOMYCIN A FERMENTATIONBACKGROUND OF THE INVENTION This invention relates to the production ofthe antibiotic called carbomycin A and, in particular, to the useofB-methylcrotonic acid, or corresponding derivatives, in its productionby fermentation.

Carbomycin A is an antibiotic with important antibacterial propertiesand wide clinical application in the treatment of bacterial infections.The use of an antibiotic, particularly on a large scale, requires thatit can be cheaply produced in large quantities.

In general terms, the media used in microbial ferrnentations include asource of nitrogen, a source of carbon and nutrient salts. Although lesswell understood, important additions include organic compounds that actas growth-promoting or potency-enhancing substances.

Organic compounds that enhance antibiotic fermentation yields may do soby direct incorporation into the antibiotic molecule or the metabolicdegradative fragments may provide additional building blocks for themetabolic synthesis of the antibiotic. The precursor has been applied tosuch substances as phenylacetic acid and other monosubstituted aceticacid derivatives which, when added to the penicillin fermentation, areincorporated directly as the side-chain moiety into the penicillinmolecule. It has been found, surprisingly, that [3 methylcrotonic acidmarkedly enhances the yield of carbomycin A when added to thefermentation medium. The precise physiological role of B-methylcrotonicacid is not clear. Microbial cell production is not increased, nor isthere evidence that it is incorporated directly into the carbomycin Amolecule. A more likely explanation is that B-methylcrotonic acid isbroken down during the fermentation process into smaller fragmentswhich, during the reassembly process, play an important role in thebiosynthesis of carbomycin A. Some supportive evidence for the indirectenhancing effect of B- methylcrotonic acid is provided by theobservation that other organic compounds such as sorbic acid, aandfiangelicalactone, isoprene, geraniol and crotyl alcohol also stimulateincreased carbomycin A when added to the fermentation medium. However,the magnitude of this effect does not approach that provided by additionof 62 methylcrotonic acid.

SUMMARY OF THE INVENTION In general, this invention embodies a processfor increasing the carbomycin A fermentation yields by the addition of Bmethylcrotonic acid, or derivatives thereof, to the fermentation media.

DETAILED DESCRIPTION OF THE INVENTION A preferred process for theproduction of carbomycin A (US. Pat. No. 2,796,379 to F. W. Tanner, Jr.,et al., June 18, 1957) involves the cultivation of Steptomyces halstediiATCC- 13449, preferably in an aqueous nutrient medium at a temperatureof about 2430 C., and under submerged conditions with agitation andaeration. Nutrient media which are useful for this process include acarbohydrate such as sugars, starch, glycerol and corn starch; a sourceof organic nitrogen such as that contained in casein, soybean meal,peanut meal, wheat gluten, cotton seed meal, lactalbumin, tryptone andenzymatic digest of casein. The use of enzymatic digest of casein as anitrogen source is preferred. Additional growth substances are providedby the addition of such substances as distillers'solubles, yeastextract, molasses or fermentation residues, as well as mineral saltssuch as sodium chloride, potassium phosphate, sodium nitrate andmagnesium sulfate. A soluble, nongrowth inhibiting, nonionic (poly)oxyethylene glycol ether surfaceactive agent, in an amount to provide aconcentration of about 0.2-4 percent is included in the fermentationprocess. If excessive foaming is encountered during the fermentation,antifoaming agents, such as vegetable oils, may be added to thefermentation medium. The pH of the fermentation tends to remain ratherconstant, but, if variations are encountered, a buffering agent such ascalcium carbonate may also be added to the medium.

It should be further observed that where alkali salts are specified,most salts will fulfill this definition at the levels and pH that areemployed. Typical alkali salts which may be employed include the sodium,potassium, ammonium, lithium, calcium and barium salts. These compoundsare generally best added at the time of medium makeup. However, asterile solution or suspension of the compound may be added up to 24hours after the addition of the inoculum.

An embodiment of this invention is the addition of )3- methylcrotonicacid, alkali salts, or a derivative thereof, to the fermentation mediaat a concentration of about 0.5 to 5.0 millimoles, preferably 2.0 2.5millimoles, per liter. It is understood that B-methylcrotonic acid, or asource of B-methylcrotonic acid, comprises derivatives such as esters,amides, nitriles or other compounds which are readily converted to therespective acid or salts either by the fermentation micro-organism or byreaction with the fermentation media.

inoculum for the preparation of antibiotic carbomycin A by the growth ofa strain of S. halstedii may be obtained by employing growth from slantsof such media as Emersons agar or beef lactose. The growth may be usedto inoculate either shaken flasks or inoculum tanks for submergedgrowth, or, alternatively, the inoculum tanks may be seeded from theshaken flasks. The growth of the micro-organism usually reaches itsmaximum in about 2 to 3 days. However, variations in the equipment used,the rate of aeration, rate of stirring, etc., may affect the speed withwhich the maximum activity is reached. In general, about 24 hours to 4days is the desirable period for producing the antibiotic. Aeration ofthe medium in tanks for submerged growth is maintained at the rate ofabout onehalf to two volumes of free air per volume of broth per minute.Agitation may be maintained by suitable types of agitators generallyfamiliar to those in the fermentation industry. Aseptic conditions, ofcourse, must be maintained throughout the preparation and transfer ofthe inoculum and the growth period of the micro-organism.

Recovery of the antibiotic is accomplished by means generally familiarto those skilled in the art, such as extraction, precipitation and theuse of strong cation exchange resins.

The present invention embraces not only the use of the herein describedorganism but also of mutants thereof produced by subjecting the organismto such measures as X- radiation, ultraviolet radiation, nitrogenmustard and the like.

The following examples are merely illustrative and are not intended tolimit the invention, the scope of which is defined by the appendedclaims.

EXAMPLE I An inoculum is prepared, using a growth medium having thefollowing composition:

Grams/liter Cerelose 15.0 Soy flour 30.0 MgSO,-7H,O 1.0 Calciumcarbonate 10.0

Grams/titer Enzose -081 (sugar concentrated, Corn Products, Inc.) 25.0ml. Beet molasses 22.5 Wheat Starch 5.0 Cerelose 15.0 Casein Digest (5percent) 400 ml. MnSo -H,O 1.5 MgSO 7H,O 1.25 CUSO"SH! 0 0.20B-methylcrotonic acid 0.20 (2.0 mM) EXAMPLE It The process of example 1is repeated with 0.25 grams (2.5

mM) of B-methylcrotonic acid per liter in place of 0.20 grams per liter,with comparable results.

EXAMPLE Ill The process of example 1 is repeated with theB-methylcrotonic acid replaced by 0.23 grams (2.0 mM) per liter of themethyl ester of B-methylcrotonic acid, with comparable results.

EXAMPLE IV The process of example 1 is repeated with the,B-methylcrotonic acid replaced by 0.32 grams (2.5 mM) per liter of theethyl ester of fi-methylcrotonic acid, with comparable results.

EXAMPLE V The process of example 1 is repeated with the B-methylcrotonicacid replaced, at a level of 2.0 mM per liter, of the potassium, sodium,and calcium salts of B-methylcrotonic acid, with comparable results.

EXAMPLE VI The inoculum of example 1 is used to inoculate a nutrientmedium having the following composition:

The pH of the medium is adjusted to 7.0 with potassium hydroxide.One-half ml. Antifoam A (Dow Chemical Company) and 20 ml. Tergitol NP-35(Union Carbide) are added per liter of medium, and the mixture issterilized.

A 10 percent aliquot of the inoculum of example 1 is transferred underaseptic conditions into the above nutrient medium. After agitation andaeration for two days, the broth potency is comparable to that ofexample 1.

EXAMPLE V1] The process of example V1 is repeated with 0.25 grams (2.5

mM) per liter of B-methylcrotonic acid in place of 0.20 grams per liter,with comparable results.

EXAMPLE VIII The process of example V1 is repeated with 0.05 grams (0.5mM) per liter of B-methylcrotonic acid in place of 0.20 grams per liter,with comparable results.

EXAMPLE IX The process of example V1 is repeated with 0.5 grams (5.0 mM)per liter of fl-methylcrotonic acid in place of 0.20 grams per liter,with comparable results.

EXAMPLE X The process of example V1 is repeated with 0.20 grams 2.0 mM)per liter of the amide of B-methylcrotonic acid in place offi-methylcrotonic acid, with comparable results.

I claim:

1. In the process for the production of carbomycin A by fermentation ofStreptomyces halstedii the improvement which comprises conducting thefermentation in the presence of B- methylcrotonic acid, its alkalisalts, or a derivative thereof which is readily converted to the saidacid or salts either by the fermentation micro-organism or by reactionwit the fermentation media in an amount to provide a concentration ofabout 0.50 5.0 millimoles per liter.

2. The process of claim 1 wherein said derivative is the methyl ester ofB-methylcrotonic acid.

3. The process of claim 1 wherein said derivative is the ethyl ester ofB-methylcrotonic acid.

4. The process of claim 1 wherein said salt is the sodium, potassium, orcalcium salt of B-methylcrotonic acid.

5. The process of claim l wherein said derivative is the amide ofB-methylcrotonic acid.

6. 1n the process for the production of carbomycin A by the cultivationof Streptomyces halstedii ATCC-l3449 in an aqueous, nutrient mediumunder submerged, aerobic conditions, the improvement which comprises theaddition to said medium of B-methylcrotonic acid in an amount to providea concentration of about 2.0 2.5 millimoles per liter.

2. The process of claim 1 wherein said derivative is the methyl ester ofBeta -methylcrotonic acid.
 3. The process of claim 1 wherein saidderivative is the ethyl ester of Beta -methylcrotonic acid.
 4. Theprocess of claim 1 wherein said salt is the sodium, potassium, orcalcium salt of Beta -methylcrotonic acid.
 5. The process of claim 1wherein said derivative is the amide of Beta -methylcrotonic acid.
 6. Inthe process for the production of carbomycin A by the cultivation ofStreptomyces halstedii ATCC-13449 in an aqueous, nutrient medium undersubmerged, aerobic conditions, the improvement which comprises theaddition to said medium of Beta -methylcrotonic. acid in an amount toprovide a concentration of about 2.0 -2.5 millimoles per liter.